EXERCISE 1

Microscopy
Name: Score:
Section & Group Number: Date submitted:

INTRODUCTION

The development of microscopes has allowed humans to discover and study microorganisms such as
bacteria, fungi, algae and protozoa which are too small to be seen by the naked eye. The use of these
instruments allows objects to be magnified a hundred or thousand times.

There are two general types of microscopes which mainly depend on the illuminating source and
focusing aide in order to visualize the specimen or object: the light microscope which utilizes visible light
waves and lenses, and the electron microscope which uses electron beams and electromagnets or
magnetic fields.

The type of microscope usually used in a typical laboratory setting is a light compound
microscope because it employs two lenses, the objective and ocular. It can magnify objects twice or thrice its
actual size as compared to a simple microscope. On the other hand, a simple microscope employs only a
single lens and magnifies up to 300X only.

OBJECTIVES

At the end of this exercise, the student should be able to:

a. identify and state each function of the parts of the compound microscope,
b. handle the microscope properly,
c. differentiate the two types of microscopes,
d. explain how images are formed in a microscope, and
e. determine the properties of microscope objectives.

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Activity 1. Examine and LABEL the microscope and give the FUNCTION of each of the parts.

Figure 1. Compound microscope: Parts and Functions

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Activity 1. Examine and LABEL the microscope and give the FUNCTION of each of the parts

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Table 1. The Properties of Microscope Objectives
Objectives
Property Scanning Low Power High Power Oil Immersion

Magnification 4x 10x 40-45x 90-100x

Numerical
Aperture 0.10 0.25 0.55-0.65 1.25-1.4

Working distance 17-20mm 4-8mm 0.5-0.7mm 0.1mm

Approximate
Focal length 40mm 16mm 4mm 1.8-2.0mm

Activity 1.2: Viewing an object with the compound light microscope

Materials:
● Compound light microscope
● Newspaper page - cut out “letter e”
● Scissors
● Forceps
● Beaker with water and dropper
● Clean slide and cover slip
● Paper towel

1. Obtain a prepared slide of the letter “e”.

a. Find a word containing a lowercase letter in a newspaper. Use scissors to
carefully cut out a small piece of paper that includes the letter e.
b. Pick up the piece containing the letter e with forceps and place it on the center of
a clean microscope slide.
c. Place a drop of water on the specimen
d. Place the coverslip on the slide. Put one edge of the coverslip into the drop of
water, and then slowly lower the coverslip on the specimen.
e. Remove any air bubbles from under the coverslip by gently tapping the coverslip.
f. Dry any excess water before placing the slide on the microscope stage for viewing.

2. Observe it under scanning power. Use the coarse adjustment knob to bring the specimen
into focus. Always begin focusing on an object by using the scanning objective (lowest
power). Diagram what you see below.

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 3. Compare the orientation of the “letter e” as seen with the microscope with the orientation
seen with the unaided eye. Do they appear the same in orientation? If not, what do you think
the reason is?

4. Diagram the letter “e” as seen in low power mode.

5. Switch to low power mode and observe the “letter e”. How does the letter “e”
appear different under low power as opposed to scanning power?

6. Now switch to high power. How does the letter e appear now? Diagram the “letter e” as
seen under high power below.

7. When you move the slide from right to left, how does it appear to move under
the microscope?

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 8. When you move the slide toward you, how does the letter e appear to move
under the microscope?

Guide Questions

1. How is an image formed in a light microscope?

When a direct or undeviated light from a specimen is projected by the objective, it is spread
evenly across the entire image plane at the diaphragm of the eyepiece. The light diffracted by the
specimen is brought to focus at various localized sites on the same image plane, and there the
diffracted light causes destructive interference. A consequence is the reduction in light intensity
resulting in dark areas. These patterns of light and dark are what we recognize as an image of the
specimen. Since our eyes are sensitive to variations in brightness, the image then becomes a more or
less faithful reconstitution of the original specimen.

2. Which objective has the shortest working distance? smallest depth of field? largest field of
view?

High magnification objective lenses are generally 100x. Thus, the objective lens with the
smallest working distance is 100x. Moreover, between 4x, 10x and 40x, 40x has the shortest depth of
focus while 4x has the largest field of view. The smaller lens means there is more available space to
see under the microscope. The 40x lens represents a higher magnification which means a smaller
available space to view.

3. What is the function of oil when using the oil immersion objective?

The oil allows the objective to collect more light to form an image due to its refractive index.
Therefore, the main purpose of using immersion media like oil, which has the same refractive index as
glass, is to minimize light refraction by eliminating the refractive index differences that are present in
the optical system. The medium increases the numerical aperture of the objective, indicating it gathers
more light and can create brighter images.

4. Why should we make sure that a wet mount does not have air bubbles when preparing it?

Wet mounts should ideally have no air bubbles because we may have trouble distinguishing the bubbles from
the specimen when looking under the microscope. The presence of bubbles can also keep live organisms from
moving freely, may lower the viewing resolution, and air bubbles in wet mounts may appear to have a dark ring
surrounding their edges underneath the microscope making the specimen harder to view.

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 5. What is the benefit of preparing a good wet mount?

A wet mount is made by placing a fluid solution on a slide, suspending a specimen in a solution, and
then covering the specimen and the solution with a cover slide. Preparing a good wet mount helps to
increase the specimen’s translucency and to make it easier to stain.

6. What are the different types of microscopy? Give five. Give its functions and mention one
advantage and disadvantage for each.

There are different types of microscopes and each of these has different purposes of use. These five types
of microscopes are: simple microscope, compound microscope, electron microscope, stereomicroscope,
and scanning probe microscope.

Simple microscope

Simple microscope functions the same way as the magnifying glass. It is used to see the magnified image
of an object and is made of a single convex lens with a short focal length, which magnifies the object
through angular magnification, thus producing an erect virtual image of the object near the lens.
The advantage of using simple microscope includes easy to operate as they are simple to set up and can be
operated by anyone with minimal training and knowledge. However, the main disadvantage of using a
simple microscope is that have a very low magnification cap which will make object become blurry and
hard to identify.

Compound Microscope

Compound microscope is a type of optical microscope that is used for obtaining a high-resolution image of
smaller specimens such as blood cells. Compound microscope is very easy to use and handle and it allows you
to view live samples of specimen. However, the magnifying power of a compound microscope is limited to
2000 times and certain specimens such as viruses, atoms, and molecules cannot be viewed with it.

Electron Microscope

Electron microscope uses a beam of electrons and their wave-like characteristics to magnify an object's image.
Electron Microscopes (EMs) function like their optical counterparts except that they use a focused beam of
electrons instead of photons to "image" the specimen and gain information as to its structure and composition.
The very first advantage is its great magnification but the disadvantage of this type of microscope is that it is
large, and extremely sensitive to vibration and external magnetic fields.

Stereo Microscopes

Stereo microscopes are used to look at a variety of samples that you would be able to hold in your hand. A
stereo microscope provides a 3D image or "stereo" image and typically will provide magnification between 10x
- 40x. The main advantage of stereo microscopes is that they can examine opaque specimens and provide a 3D
view of the sample however it’s disadvantage is that it is not applicable for viewing tissue structures, bacteria,
and viruses.

Scanning Probe Microscope

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Scanning probe microscopy is used to create images of nanoscale surfaces and structures or manipulate atoms
to move them in specific patterns. It involves a physical probe that scans over the surface of a specimen
gathering data that is used to generate the image or manipulate the atoms. Scanning Probe Microscopy provides
researchers with a larger variety of specimen observation environments using the same microscope and
specimen reducing the time required to prepare and study specimens. Unfortunately, one of the downsides of
scanning probe microscopes is that images are produced in black and white or grayscale which can in some
circumstances exaggerate a specimen’s actual shape or size.

7. Cite some advantages and disadvantages when using the light microscope and electron
microscope.

•Advantages of light microscope include but not limited to:

1. relatively easy to use as they are small and lightweight

2. offer high levels of observational quality
3. They are unaffected by electromagnetic fields
4. Allows you to observe living organisms
5. It can use fluorescent lights to display sample visually

•Disadvantages of light microscope include but not limited to:

1. they do not magnify at the same level as other options.

2. They have a lower resolution and cannot provide three-dimensional renderings.
3. They cannot operate in darkness
4. They can come with very low magnification caps
5. They make it challenging to view living internal structures

•Advantages of electron microscope include but not limited to:

1. they have a high resolution and high magnification

2. they are use in Improvement in Scientific Technology
3. they have a large depth of field which allows more of a specimen to be in focus
4. they are helpful in viewing intricate details of a specimen
5. have many technological and industrial applications, such as semiconductor inspection

•Disadvantages of electron microscope include but not limited to:

1. they are expensive, large and not portable

2. they cannot display living specimens in natural color
3. only dead specimen can be used
4. lots of training required to use them
5. extremely sensitive to vibration and external magnetic fields.

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